Skip to main content
Book cover

Mammalian Cell Engineering pp 3–14Cite as

Generation of CAR-T Cells by Lentiviral Transduction

Part of the Methods in Molecular Biology book series (MIMB,volume 2312)

Abstract

CAR-T cell therapy is one of the most successful cell-based therapies. T cells are the most common cells to be genetically modified for cancer therapy, not only because T cells have cytotoxicity but also because they are easily cultured ex vivo and genetically modified with viral vectors. Hence, for nonexperts, T cell engineering is an ideal starting point for mammalian cell engineering or for development of therapeutics. Here, we have described a basic procedure for lentiviral transduction of human primary T cells to generate a CAR-T cell and assays to confirm CAR expression and function.

Key words

  • CAR-T cell
  • Lentivirus vector
  • Dynabeads
  • PEG precipitation
  • Retronectin
  • Flow cytometry
  • CAR-T cell activation assay

This is a preview of subscription content, access via your institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-0716-1441-9_1
  • Chapter length: 12 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   109.00
Price excludes VAT (USA)
  • ISBN: 978-1-0716-1441-9
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   139.99
Price excludes VAT (USA)
Hardcover Book
USD   219.99
Price excludes VAT (USA)
Learn about institutional subscriptions
Fig. 1
Fig. 2
Fig. 3
Fig. 4

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. June CH, O’Connor RS, Kawalekar OU, Ghassemi S, Milone MC (2018) CAR T cell immunotherapy for human cancer. Science 359:1361–1365

    CrossRef  CAS  Google Scholar 

  2. Grupp SA, Kalos M, Barrett D et al (2013) Chimeric antigen receptor–modified T cells for acute lymphoid leukemia. N Engl J Med 368:1509–1518

    CrossRef  CAS  Google Scholar 

  3. Maude SL, Frey N, Shaw PA et al (2014) Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 371:1507–1517

    CrossRef  Google Scholar 

  4. Search of: CAR-T cell—List Results—ClinicalTrials.gov. https://clinicaltrials.gov/ct2/results?cond=&term=CAR-T+cell&cntry=&state=&city=&dist=. Accessed 14 May 2020

  5. MacKay M, Afshinnekoo E, Rub J et al (2020) The therapeutic landscape for cells engineered with chimeric antigen receptors. Nat Biotechnol 38:233–244

    CrossRef  CAS  Google Scholar 

  6. Morsut L, Roybal KT, Xiong X, Gordley RM, Coyle SM, Thomson M, Lim WA (2016) Engineering customized cell sensing and response behaviors using synthetic notch receptors. Cell 164:780–791

    CrossRef  CAS  Google Scholar 

  7. Roybal KT, Williams JZ, Morsut L, Rupp LJ, Kolinko I, Choe JH, Walker WJ, McNally KA, Lim WA (2016) Engineering T cells with customized therapeutic response programs using synthetic notch receptors. Cell 167:419–432.e16

    CrossRef  CAS  Google Scholar 

  8. Cho JH, Okuma A, Al-Rubaye D, Intisar E, Junghans RP, Wong WW (2018) Engineering Axl specific CAR and SynNotch receptor for cancer therapy. Sci Rep 8:1–8

    Google Scholar 

  9. Cho JH, Collins JJ, Wong WW (2018) Universal chimeric antigen receptors for multiplexed and logical control of T cell responses. Cell 173:1426–1438.e11

    CrossRef  CAS  Google Scholar 

  10. Cho JH, Okuma A, Sofjan K, Lee S, Collins JJ, Wong WW (2021) Engineering advanced logic and distributed computing in human CAR immune cells. Nat Commun. 12:792

    Google Scholar 

  11. Milone MC, O’Doherty U (2018) Clinical use of lentiviral vectors. Leukemia 32:1529–1541

    CrossRef  CAS  Google Scholar 

  12. Marcucci KT, Jadlowsky JK, Hwang W-T et al (2018) Retroviral and lentiviral safety analysis of gene-modified T cell products and infused HIV and oncology patients. Mol Ther 26:269–279

    CrossRef  CAS  Google Scholar 

  13. Long AH, Haso WM, Shern JF et al (2015) 4-1BB Costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat Med 21:581–590

    CrossRef  CAS  Google Scholar 

  14. Broad Institute Cancer Cell Line Encyclopedia (CCLE). https://portals.broadinstitute.org/ccle. Accessed 14 May 2020

Download references

Acknowledgments

I thank Wilson W. Wong (Boston University) and Jang Hwan Cho (Broad Institute, MIT) for kindly sharing their T cell transduction procedure and accepting to use the data in this paper. I also thank Satoshi Mimura (Hitachi Ltd.) for helping to optimize this procedure. This work was supported by Leading Initiative for Excellent Young Researchers grant from Japan Society for the Promotion of Science.

Author information

Authors and Affiliations

  1. Center for Exploratory Research, Research & Development Group, Hitachi Ltd., Chiyoda City, Tokyo, Japan

    Atsushi Okuma

Authors
  1. Atsushi Okuma
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Atsushi Okuma .

Editor information

Editors and Affiliations

  1. Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Ryosuke Kojima

Rights and permissions

Reprints and Permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Verify currency and authenticity via CrossMark

Cite this protocol

Okuma, A. (2021). Generation of CAR-T Cells by Lentiviral Transduction. In: Kojima, R. (eds) Mammalian Cell Engineering. Methods in Molecular Biology, vol 2312. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1441-9_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1441-9_1

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1440-2

  • Online ISBN: 978-1-0716-1441-9

  • eBook Packages: Springer Protocols